Fig 7. Synthetic TALE transcription factors targeting the TEP1 promoter.

a. Schematic representation of the ~3Kb TEP1 promoter (not to scale). Shown are potential NF-κB sites (GGGRNNYYCC) (light grey boxes) and potential TATA boxes (dark grey boxes). The minimal promoter (MP) shown to be active in cells is indicated by a black line. Numbers on the promoter indicate the distance from the ATG. White boxes represent the binding position of each designed TALE. b. Luciferase assay in S2 cells showing the effect of TALEs on the minimal TEP1 promoter. Different TALE constructs all carrying a VP16 activation domain and placed under the Drosophila actin5c promoter were co-transfected into S2 cells together with a firefly luciferase reporter gene under control of the minimal TEP1 promoter. A plasmid carrying GFP placed under the same promoter as TALEs was used as reference for basal promoter activity. The TEP1 promoter was further induced by addition of heat killed E. coli to the cells. Luciferase activity was normalized to the basal promoter activity without TALEs. c. qRT-PCR to assess the level of TALE and TEP1 mRNA in transgenic mosquitoes expressing Vg-TAL₆ comparing functional TALE with VP16 activation domain (Vg-TAL₆^vp16) and control line expressing the same TALE without activation domain (Vg-TAL₆^ΔAD), with or without induction of the Vg promoter by a blood meal 24h prior to mRNA extraction. d. qRT-PCR of transgenic mosquitoes constitutively expressing Lp-TAL₆ or Lp-TAL₀ or both, showing the level of TEP1 mRNA. Levels of mRNA in each sample are normalized to wt and to the housekeeping reference gene RPL19. e Western blot analysis of transgenic mosquitoes constitutively expressing Lp-TAL₆ or Lp-TAL₀ or both. Wild type mosquitoes are used as controls. Shown are the bands for the full length and the cleaved forms of TEP1 as well as PPO2 as loading control. 2 samples for each mosquito line are presented to show sample variation. f. Quantification by densitometry of the TEP1 bands in (e). g. P. berghei infection of Vg-TAL^vp16 transgenic mosquitoes. h. P. berghei Infection of transgenic mosquitoes constitutively expressing TAL₀, TAL₆ or both under the Lp promoter. In all infections, the empty docking line (wt^X1) serves as control. Live oocysts (green circles) in each midgut are counted 7 days after infection. Number of oocysts and infection data are in S3 Table.